Transfection of mammalian cells is a procedure whereby a molecule is placed into a cell by passing it through the cell""s outer membrane without perturbing the viability of the cell. Molecules such as DNA, RNA, and proteins are often the transfected material.
Generally, the transfectant will not passively pass through the intact cell membrane. Its passage is aided by transient disruption of the membrane. Four methods are in common use.
Gene gun wherein the transfectant is first bound to a solid particle such as graphite or gold and shot at high velocity into the cell.
Liposome mediated transfection wherein a liposome complex is formed between lipid and transfectant. The liposomes interact with the cell membrane facilitating the passage of the transfectant into the cell.
Calcium phosphate, that chelates DNA and form a precipitate that is internalised.
Electroporation mediated transfection wherein an electrical field pulse is applied across the cell membrane which causes transient pore formation in the cell membrane. This enables the transfecting molecule to pass through the pore into the cell.
A prerequisite for all the above methods is that sufficient cells are present for transfection. This is achieved by prior expansion of cells by culture. The Gene gun is not usually a method of choice, as it is inefficient and requires that the cells are grown adhered to a surface. Liposome and electroporation mediated transfection are more efficient than the Gene gun. Liposome mediated transfection has been more popular than electroporation mediated transfection. This is partly due to the fact that liposome mediated transfection can be carried out on cells growing in solution and cells grown adhered to a surface. Electroporation mediated transfection is more efficient than liposome mediated transfection and is generally carried out on cells grown in solution. With the advent of electrophorus membranes (e.g., Corning) which allow electroporation of adherently grown cells, this imbalance is likely to be redressed. In both instances, standard cell culture media is a poor media for transfection. Therefore, culture media must be replaced with a suitable transfection solution prior to transfection.
The majority of cultured cells used in industry and academia are used for transient or stable (permanent integration of the transfected molecule) transfection studies. These cells lines are available through organizations such as the ETACC and ATCC. Cells lines are obtained as a frozen aliquot and clonally expanded by the purchaser. Once expanded, the cells are generally kept as a growing culture and as a frozen stock. This growing culture is the source of cells for transfection. Cells are generally frozen in a cocktail of enriched culture media and cryoprotectant. The most common cryoprotectant used is dimethyl sulfoxide (DMSO). However, glycerol, sucrose, trahalose and proline and others are sometimes used. Cryoprotectants are thought to aid cell survival by preventing ice nucleation in cells, which is a contributory factor to cell rupture upon freezing, while the culture media provides nutrients. The multiple factors involved in successful cryo-preservation of Animal cells have been extensively reviewed by Doyle et. al. 1988.
The generally used procedure for transfection is as follows:
(1) Culture cells to produce sufficient cells for transfection. This step can take several days.
(2) Transfect cells.
(3) Study transfected cells.
The disadvantages of this procedure is that pre-culture of cells for transfection requires an excessive amount of time and resources. Moreover, the requirement for transfection to be repeated each time an experiment is undertaken may lead to variability in transfection.
The instant invention is a cryoprotectant for electroporation ready cells comprising molecules that protect cells during the freeze thaw cycle prior to electroporation.
It is a high efficiency electroporation buffer containing antioxidants and other critical molecules that help in the regeneration process after electroporation.
The cryoprotectant/electroporation buffer comprises the cold protection of culture media with cryoprotectant and the transfection capabilities of electroporation media.
The dual purpose buffer combines a cell-freezing medium and a high efficiency mammalian cell transfection buffer.
The buffer is selected from a range of cryoprotectants as described in Stacey, 1997, Stange and Mitzner, 1996, Grout et al 1990 and Doyle et al 1988, that include DMSO, glycerol, sucrose, trehalose, and protein.
Preferred buffer is DMSO, glycerol or sucrose.
The buffer is useful simultaneously and directly as a high efficiency electroporation transfection buffer and as a buffer to freeze cells for long-term storage.
The invention is further a method of buffering transfected material comprising:
a) freezing the material in electroporation/cryoprotection media,
b) resuspending the material in water,
c) resuscitating by defrosting at room temperature for about 1 minute and then 37xc2x0 C. for xcx9c2 minutes, depending on cell type and apparatus available.
d) mixing an aliquot of material with cells and placing them in an electroporation cell followed by transfecting the material in a culture dish
In the invention, Cytomix, an intracellular ionic strength- and pH-mimicking buffer composed of 120 mM KCl; 0.15 mM CaCl2; 10 mM K2HPO4/KH2PO4, pH 7.6; 25 mM Hepes, pH 7.6; 2 mM EGTA, pH 7.6; and 5 mM MgCl2; pH adjusted with KOH, with added water and DMSO to final volume is resuspended and aliquoted into cryovials, wrapped and frozen to xe2x88x9220xc2x0 C. over xcx9c5 hours and then xe2x88x9270xc2x0 C. overnight, deep freezing in nitrogen for xcx9c5 days wherein the latter stage of freezing depends on the sophistication of the available apparatus and has an impact on the successful preservation.
The instant invention eliminates the need for repetitive pre-culture and therefore has the following advantages over the art. Experiments are initiated at the transfection step, significantly reducing the time and resources required greatly. The number of experimental steps is reduced and so is the likelihood of infection of cells. With all of the transfected cells from the same stock, there is a reduction of intra- and inter-experimental variation. This invention allows one to do a different experiment each day; one is not confined to one available batch.
In the instant invention, the need to pre-culture cells prior to transfection is eliminated by producing a frozen cell stock. This cell stock has the following characteristics: each aliquot has sufficient cells for a transfection; cells are viable when resuscitated; and cells are capable of immediate transfection upon resuscitation. The transfection technique used is already established, e.g., liposome transfection or electroporation. For each aliquot to have sufficient cells for a transfection, cells must first be cultured in sufficient quantities for stocks to be made. For the cells to be viable when resuscitated and be capable of immediate transfection upon resuscitation, a buffer which has a dual function is required. The buffer must function as a cryoprotectant and a transfection buffer. This buffer is the object of the instant invention.
The invention is a cryoprotectant/electroporation buffer useful simultaneously and directly as a high efficiency electroporation transfection buffer and as a buffer to freeze cells for long-term storage. This is achieved by combining the cold protection of culture media with cryoprotectant and the transfection capabilities of electroporation media. This alleviates the need for continuous cell culture and repeated transfections due to transfection variability of current methods.
Cytomix (Knight and Scrutton 1986) also sold as Optimix (marketed by EquiBio) is an intracellular ionic strength mimicking buffer. This buffer protects against osmotic rupture as it has the same osmotic strength as the cell contents. It also contains additional salts and other critical molecules, including antioxidants that help in the regeneration process during and following destabilization caused by the electrical discharge through the cell (Tsong 1991). Cytomix has the following composition; 120 mM KCl, 0.15 mM CaCl2; 10 mM K2HPO4/KH2PO4, pH 7.6; 25 mM Hepes, pH 7.6; 2 mM EGTA, pH 7.6; 5 mM MgCl2; pH adjusted with KOH) with ATP (pH 7.6 adjusted with KOH 2 mM) and Glutathione (5 mM) added just before electroporation (Van den Hoff et al., Nucleic Acid Research, 2902 Vol 20 No. 11 and Knight D. E. and Scrutton M. C., Biochem J., 1986;234:497-506). This buffer offers good transfection efficiency and is thought to prevent leakage of the cytoplasmic components, protect membranes against oxidation, and facilitate resealing of pores. However, it does not offer good protection for freezing of mammalian cells.
Dimethyl Sulfoxide is used as a model cryoprotectant. Addition of DMSO to the above electroporation buffer results in a novel dual purpose buffer which is used directly as a cell freezing buffer and a high efficiency electroporation transfection buffer.
A detailed protocol follows. The salient points of the experiment are outlined below.
(1) Cos-7 cells were cultured in media consisting of DMEM (500 ml Gibco Cat. No. 31966-021), FBS (50 ml Gibco Cat No. 10100-0155) and pen/strep (5 ml Gibco Cat No. 15070-022) at 37xc2x0 C. according to protocol in 6xc3x97T175 flasks until 70-80% confluent.
(2) Cos-7 cells were detached from the flask with trypsine (Gibco Cat. No. 45300-019) and peletted at 200 g for 5 minutes at room temperature. Cells were washed twice in 50 ml of Optimem-1 (Gibco Cat. No. 51985-026) at room temperature then resuspended in EquiBio buffer A (cytomix buffer-ATP-Glutathione).
(3) Cos-7 cells were split into two aliquots of 10xc3x97106 cells and half transformed immediately and half frozen in 2xc3x970.7 ml aliquots.
(4) To EquiBio buffer B (2xc3x97cytomix buffer) was added ATP and Glutathione. Cells (10xc3x97106) was resuspended in a solution of 800 xcexcl of the above with 800 xcexcl of H2O with (60 xcexcg of pGL3-Control, encoding luciferase gene) according to the protocol and 800 xcexcl placed in a 4 mm electroporation cell (Flowgen E5-0100).
(5) Cells were immediately transformed at 250V, 1050 xcexcF, and infinite resistance.
(6) An aliquot (50 xcexcL) was plated out in 6-well plates and cultured overnight.
(7) Cos-7 cells were harvested in a similar manner to above and luciferase activity measured.
(8) The remaining Cos-7 cells were frozen in electroporation/cryoprotection media as follows:
To 2xc3x97Optimix (above) was added H2O and 5% DMSO (75 xcexcL) to a final volume of 1400 xcexcL. Cells (10xc3x97106) were resuspended in the above and 2xc3x970.7 ml was aliquoted into cryovials. Cells were wrapped in cotton wool and frozen to xe2x88x9220xc2x0 C. over a time period of about 5 hours and then xe2x88x9270xc2x0 C. overnight. Cells were deep frozen in liquid nitrogen for 5 days.
(9) DNA (60 xcexcg) was resuspended in 200 xcexcl of H2O.
(10) Frozen Cos-7 cells were resuscitated by defrosting at room temperature for 1 minute and then 37xc2x0 C. for 2 minutes.
(11) A 100 xcexcl aliquot of DNA was quickly mixed with cells (700 xcexcl) and then placed in an electroporation cell. Cells were transfected as above in 6-well culture dish.
Cells were observed 1 hour and 18 to 24 hours after transfection. In both cases, transfected cells had a similar morphology and bedded down on the culture plates in a similar manner.
Luciferase activity was measured 18 to 24 hours after transfection as described above. Luciferase activity of non-frozen Cos-7:
(1) 586.2
(2) 582.8
(3) 591.8
(4) 573.8
(5) 559.4
(6) 572.3 Average=577.7
These results show that cells frozen in electroporation/cryoprotection buffer are capable of being viable after being frozen in liquid nitrogen and are capable of being transfected by electroporation. The non-frozen cells are more electroporationally competent than the frozen cells due to several possible factors: Not all cells that are frozen are viable and addition of DMSO possibly reducing the electroporation efficiency of the buffer.
A comparison of luciferase activity in the non-frozen and frozen transfected cells indicated that frozen cells and non-frozen cells have luciferase activity. This shows that both cell types have been transfected. Previous studies indicated that non-transfected cells do not have luciferase activity.
Non-frozen cells have three times more luciferase activity than frozen cells. This indicates that there was less luciferase activity in the frozen cells. This could be due to less viable cells present after resuscitation for transfection or to a decrease in the electroporation efficiency. An active method of cell freezing coupled to cryoprotection optimization will improve transfection efficiency.
Cell culture, harvesting, transfection, luciferase activity measurements and cell freezing were standard laboratory techniques. With regards to cell freezing, the method used was determined by the availability of equipment. A more sophisticated active method would produce a higher titre of viable cells (see Upstream Processes: Equipment and Techniques, 1988; Doyle A. et al., Cryopreservation of Animal Cells, 19 :1-17). The protocol for cell resuscitation was generally known. Other more sophisticated protocols can be used.
For efficient transfection, it is important to have a solution of transfectant in this example DNA. The DNA was dissolved in water, but transfectants can be resuspended in most solution which allows solubilization.